Polyploidy, the increase of diploid DNA content per cell, occurs in a variety of cells, including megakaryocytes (MKs), where it is achieved by an endomitotic cell cycle. The degree of MK ploidy influences platelet level and quality. In the search for regulators of MK endomitosis, we found that cyclin D3 is the predominant D-type cyclin in this lineage and that its upregulation, either ectopically or by thrombopoietin treatment, increases MK ploidy level in vivo. Recently, it was reported that an in vivo knock out of cyclin E, a known target of cyclin D3, significantly diminishes MK ploidy level. This is strikingly different from the normal development of other lineages and organs in these mice. Based on the known ability of cyclin E to rescue cells from a resting GO phase and on our related findings, we propose the novel contention that MKs follow few endomitotic cell cycles with transition into a resting phase, and that cyclin E uniquely allows cell cycle re-entry. Polyploidizing MKs are also programmed to skip late anaphase and cytokinesis. We hypothesize that the spindle midzone at late anaphase is atypically configured and that this feature and associated changes in chromosome passenger proteins (our recent finding) are of functional significance. To enhance exploration of related mechanisms, we intend to develop a novel mouse model with MKs containing marked chromosomes and microtubules that will allow live imaging of cells. Via this approach and expression experiments, we will study chromosome and microtubule dynamics as well as the degree of continuity of endomitotic cycles. Four Specific Aims of research are proposed: 1. To examine the ability of elevated cyclin E to promote MK ploidy in vivo; 2. To explore the molecular mechanism of cyclin E requirement for MK polyploidization; 3. To generate an in vivo model of MKs with labeled chromosomes and microtubules and to study the dynamics of endomitosis in this lineage; 4. To study the process of redistribution of chromosome passenger proteins during MK endomitosis and its effect on MK ploidy level. Taken together, pursuing these aims of research should enhance our understanding of the molecular mechanisms of megakaryocyte polyploidization, a process that impacts platelet biogenesis and hence, blood hemostasis.